The chemical complexity of pharmaceutical proteins makes it difficult to produce them synthetically for medical treatments. For many of these proteins, the only practical production route is to grow bacterial cultures that have been designed to produce the protein in adequate quantities. Thus, pharmaceutical manufacturers, among others, have an active interest in developing devices and methods for the study of bacterial cultures. One technique for measuring the size and growth rate of bacterial cultures involves counting bacteria colonies using plating, which relies on the colony-forming ability of viable cells. A sample of an appropriately diluted culture is dispersed on a solid medium and the number of colonies that form is determined. Unfortunately, plating can produce inexact measurements because the culture continues to grow at an unknown rate during the period of dilution in preparation for plating. In another technique, the total number of cells can be determined microscopically by determining the number of cells per unit area in a counting chamber (a glass slide with a central depression of known depth, whose bottom is ruled into squares of known area). However, this technique is a hands-on, serial process that is prone to human error.
Counting errors may be reduced by using electronic counting devices, such as a coulter counter, which can determine the size distribution as well as the number of bacteria in a sample culture of known volume. The coulter counter relies on a pore, through which a known volume of suspension is pumped. Although the counter is rapid and accurate, it is also expensive and subject to a number of artifactual complications. Moreover, the pore through which the suspension is pumped is prone to clogging if the media and diluents are not carefully prepared.
Another technique for studying and measuring bacterial cultures involves determining the dry weight of cells in a known volume of suspension. This technique is time consuming and requires a considerable amount of sacrificial culture. As such, it is unsuitable for routine monitoring of the growth rate. Optical density has also been used to determine growth rates using cell density. However, the correlation between cell density and optical density of the culture may change during production of proteins that aggregate and form inclusion bodies.
Chemostats may also be used to study and measure bacterial cultures. These devices can maintain a constant population of bacteria in a state of active growth. This may be done by periodically substituting a fraction of a microbial culture with an equal volume of fresh, sterile, chemically defined growth medium. The influent composition may be such that the ingredients are in optimal amounts except for the growth-limiting factor, whose concentration is kept sufficiently low. At an adequate flow rate, a low concentration of the growth-limiting factor establishes itself in the growth chamber.
At sufficiently low concentrations of the growth-limiting factor the microbial growth rate is directly proportional to the concentration of the growth-limiting factor and independent of other nutrient factors, as well as bacterial metabolites. The bacterial population may automatically proceed towards a steady state of growth, where the cell density remains constant and the growth rate is sufficient to replace the cells lost in the effluent. The steady-state cell concentration may be varied by changing the dilution rate, or the concentration of the growth-limiting factor in the influent.
Data collection with conventional chemostats is not easy to automate, which makes studies and measurements of the bacterial cultures labor intensive. The devices also consume large amounts of growth medium that increase the cost of experiments, especially when costly reagents have to be used.
Another difficulty with chemostats is their tendency to form biofilms on growth-chamber walls and probe surfaces. The biofilms can start when microorganisms (e.g., bacteria from the culture) attach to a wall or probe surface during the course of chemostat operation. Once started, the biofilms are difficult to remove and may consume a significant fraction of the substrate. This may compromise the fixed biomass fundamental conservation principle of the chemostat, inducing hybrid batch/chemostat characteristics. The significance of this artifact may be magnified in laboratory scale chemostats where the surface area to volume ratio is large. Thus there remains a need for chemostat technology that suppresses or prevents biofilm growth, and consumes smaller amounts of growth medium, among other characteristics.